Marijke Stigter-van Walsum

Improved tumor targeting of anti–epidermal growth factor receptor Nanobodies through albumin binding: taking advantage of modular Nanobody technology

The ∼15-kDa variable domains of camelid heavy-chain-only antibodies (called Nanobodies) can easily be formatted as multivalent or multispecific single-chain proteins. Because of fast excretion, however, they are less suitable for therapy of cancer. In this study, we aimed for improved tumor targeting of a bivalent anti–epidermal growth factor receptor (EGFR) Nanobody (αEGFR-αEGFR) by fusion to a Nanobody unit binding to albumin (αAlb). Biodistributions of αEGFR-αEGFR, αEGFR-αEGFR-αAlb (∼50 kDa), αTNF-αTNF-αAlb (control, binding tumor necrosis factor-α), and the ∼150-kDa anti-EGFR antibody cetuximab were compared in A431 xenograft-bearing mice. The proteins were radiolabeled with 177Lu to facilitate quantification. Tumor uptake of 177Lu-αEGFR-αEGFR decreased from 5.0 ± 1.4 to 1.1 ± 0.1 %ID/g between 6 and 72 h after injection. Due to its rapid blood clearance, tumor-to-blood ratios >80 were obtained within 6 h after injection. Blood clearance became dramatically slower and tumor uptake became significantly higher by introduction of αAlb. Blood levels of αEGFR-αEGFR-αAlb were 21.2 ± 2.5, 11.9 ± 0.6, and 4.0 ± 1.4 and tumor levels were 19.4 ± 5.5, 35.2 ± 7.5, and 28.0 ± 6.8 %ID/g at 6, 24, and 72 h after injection, respectively. Tumor uptake was at least as high as for cetuximab (15.5 ± 3.9, 27.1 ± 7.9, and 25.6 ± 6.1 %ID/g) and significantly higher than for αTNF-αTNF-αAlb. αEGFR-αEGFR-αAlb showed faster and deeper tumor penetration than cetuximab. These data show that simple fusion of αEGFR and αAlb building blocks results in a bifunctional Nanobody format, which seems more favorable for therapy as far as pharmacokinetics and tumor deposition are concerned. [Mol Cancer Ther 2008;7(8):2288–97]

Long-lived positron emitters zirconium-89 and iodine-124 for scouting of therapeutic radioimmunoconjugates with PET

Antibody-PET imaging might be of value for the selection of radioimmunotherapy (RIT) candidates to confirm tumor targeting and to estimate radiation doses to tumor and normal tissues. One of the requirements to be set for such a scouting procedure is that the biodistributions of the diagnostic and therapeutic radioimmunoconjugates should be similar. In the present study we evaluated the potential of the positron emitters zirconium-89 ((89)Zr) and iodine-124 ((124)I) for this approach, as these radionuclides have a relatively long half-life that matches with the kinetics of MAbs in vivo (t(1/2) 3.27 and 4.18 days, respectively). After radiolabeling of the head and neck squamous cell carcinoma (HNSCC)-selective chimeric antibody (cMAb) U36, the biodistribution of two diagnostic (cMAb U36-N-sucDf-(89)Zr and cMAb U36-(124)I) and three therapeutic radioimmunoconjugates (cMAb U36-p-SCN-Bz-DOTA-(88)Y-with (88)Y being substitute for (90)Y, cMAb U36-(131)I, and cMAb U36-MAG3-(186)Re) was assessed in mice with HNSCC-xenografts, at 24, 48, and 72 hours after injection. Two patterns of biodistribution were observed, one pattern matching for (89)Zr- and (88)Y-labeled cMAb U36 and one pattern matching for (124)I-, (131)I-, and (186)Re-cMAb U36. The most remarkable differences between both patterns were observed for uptake in tumor and liver. Tumor uptake levels were 23.2 +/- 0.5 and 24.1 +/- 0.7%ID/g for the (89)Zr- and (88)Y-cMAb U36 and 16.0 +/- 0.8, 15.7 +/- 0.79 and 17.1 +/- 1.6%ID/g for (124)I-, (131)I-, and (186)Re-cMAb U36-conjugates, respectively, at 72 hours after injection. For liver these values were 6.9 +/- 0.8 ((89)Zr), 6.2 +/- 0.8 ((88)Y), 1.7 +/- 0.1 ((124)I), 1.6 +/- 0.1 ((131)I), and 2.3 +/- 0.1 ((186)Re), respectively. These preliminary data justify the further development of antibody-PET with (89)Zr-labeled MAbs for scouting of therapeutic doses of (90)Y-labeled MAbs. In such approach (124)I-labeled MAbs are most suitable for scouting of (131)I- and (186)Re-labeled MAbs.

Rapid visualization of human tumor xenografts through optical imaging with a near-infrared fluorescent anti-epidermal growth factor receptor nanobody.

Given that overexpression of the epidermal growth factor receptor (EGFR) is found in many types of human epithelial cancers, noninvasive molecular imaging of this receptor is of great interest. A number of studies have employed monoclonal antibodies as probes; however, their characteristic long half-life in the bloodstream has encouraged the development of smaller probes. In this study, an anti-EGFR nanobody-based probe was developed and tested in comparison with cetuximab for application in optical molecular imaging. To this aim, the anti-EGFR nanobody 7D12 and cetuximab were conjugated to the near-infrared fluorophore IRDye800CW. 7D12-IR allowed the visualization of tumors as early as 30 minutes postinjection, whereas with cetuximab-IR, no signal above background was observed at the tumor site. Quantification of the IR-conjugated proteins in the tumors revealed ≈ 17% of injected dose per gram 2 hours after injection of 7D12-IR, which was significantly higher than the tumor uptake obtained 24 hours after injection of cetuximab-IR. This difference is associated with the superior penetration and distribution of 7D12-IR within the tumor. These results demonstrate that this anti-EGFR nanobody conjugated to the NIR fluorophore has excellent properties for rapid preclinical optical imaging, which holds promise for its future use as a complementary diagnostic tool in humans.

Llama-derived Single Variable Domains (Nanobodies) Directed against Chemokine Receptor CXCR7 Reduce Head and Neck Cancer Cell Growth in Vivo

Background: The atypical chemokine receptor CXCR7 is highly expressed in various types of cancer. Results: CXCR7 Nanobodies were generated and show inhibition of β-arrestin2 signaling and secretion of angiogenic CXCL1 in vitro. Anti-CXCR7 Nanobodies reduce tumor growth by inhibiting angiogenesis. Conclusion: CXCR7 inhibition by Nanobodies inhibit head and neck tumor formation. Significance: Anti-CXCR7 therapies are potential novel treatments against head and neck cancer. The chemokine receptor CXCR7, belonging to the membrane-bound G protein-coupled receptor superfamily, is expressed in several tumor types. Inhibition of CXCR7 with either small molecules or small interference (si)RNA has shown promising therapeutic benefits in several tumor models. With the increased interest and effectiveness of biologicals inhibiting membrane-bound receptors we made use of the “Nanobody platform” to target CXCR7. Previously we showed that Nanobodies, i.e. immunoglobulin single variable domains derived from naturally occurring heavy chain-only camelids antibodies, represent new biological tools to efficiently tackle difficult drug targets such as G protein-coupled receptors. In this study we developed and characterized highly selective and potent Nanobodies against CXCR7. Interestingly, the CXCR7-targeting Nanobodies displayed antagonistic properties in contrast with previously reported CXCR7-targeting agents. Several high affinity CXCR7-specific Nanobodies potently inhibited CXCL12-induced β-arrestin2 recruitment in vitro. A wide variety of tumor biopsies was profiled, showing for the first time high expression of CXCR7 in head and neck cancer. Using a patient-derived CXCR7-expressing head and neck cancer xenograft model in nude mice, tumor growth was inhibited by CXCR7-targeting Nanobody therapy. Mechanistically, CXCR7-targeting Nanobodies did not inhibit cell cycle progression but instead reduced secretion of the angiogenic chemokine CXCL1 from head and neck cancer cells in vitro, thus acting here as inverse agonists, and subsequent angiogenesis in vivo. Hence, with this novel class of CXCR7 inhibitors, we further substantiate the therapeutic relevance of targeting CXCR7 in head and neck cancer.

Inert coupling of IRDye800CW to monoclonal antibodies for clinical optical imaging of tumor targets

BackgroundPhotoimmunodetection, in which monoclonal antibodies [mAbs] are labeled with fluorescent dyes, might have clinical potential for early detection and characterization of cancer. For this purpose, the dye should be coupled in an inert way to mAb. In this study, different equivalents of IRDye800CW, a near-infrared fluorescent dye, were coupled to 89Zr-labeled cetuximab and bevacizumab, and conjugates were evaluated in biodistribution studies. Radiolabeled mAbs were used to allow accurate quantification for assessment of the number of dye groups that can be coupled to mAbs without affecting their biological properties.Methods89Zr-cetuximab and 89Zr-bevacizumab, containing 0.5 89Zr-desferal group per mAb molecule, were incubated with 1 to 10 eq IRDye800CW at pH 8.5 for 2 h at 35°C, and 89Zr-mAb-IRDye800CW conjugates were purified by a PD10 column using 0.9% NaCl as eluent. HPLC analysis at 780 nm was used to assess conjugation efficiency. In vitro stability measurements were performed in storage buffer (0.9% NaCl or PBS) at 4°C and 37°C and human serum at 37°C. 89Zr-mAb-IRDye800CW conjugates and 89Zr-mAb conjugates (as reference) were administered to nude mice bearing A431 (cetuximab) or FaDu (bevacizumab) xenografts, and biodistribution was assessed at 24 to 72 h after injection.ResultsConjugation efficiency of IRDye800CW to 89Zr-mAbs was approximately 50%; on an average, 0.5 to 5 eq IRDye800CW was conjugated. All conjugates showed optimal immunoreactivity and were > 95% stable in storage buffer at 4°C and 37°C and human serum at 37°C for at least 96 h. In biodistribution studies with 89Zr-cetuximab-IRDye800CW, enhanced blood clearance with concomitant decreased tumor uptake and increased liver uptake was observed at 24 to 72 h post-injection when 2 or more eq of dye had been coupled to mAb. No significant alteration of biodistribution was observed 24 to 48 h after injection when 1 eq of dye had been coupled. 89Zr-bevacizumab-IRDye800CW showed a similar tendency, with an impaired biodistribution when 2 eq of dye had been coupled to mAb.ConclusionUsage of 89Zr-mAbs allows accurate quantification of the biodistribution of mAbs labeled with different equivalents of IRDye800CW. Alteration of biodistribution was observed when more than 1 eq of IRDye800CW was coupled to mAbs.

Development of Novel ADCs: Conjugation of Tubulysin Analogues to Trastuzumab Monitored by Dual Radiolabeling

Tubulysins are highly toxic tubulin-targeting agents with a narrow therapeutic window that are interesting for application in antibody-drug conjugates (ADC). For full control over drug-antibody ratio (DAR) and the effect thereof on pharmacokinetics and tumor targeting, a dual-labeling approach was developed, wherein the drug, tubulysin variants, and the antibody, the anti-HER2 monoclonal antibody (mAb) trastuzumab, are radiolabeled. (131)I-radioiodination of two synthetic tubulysin A analogues, the less potent TUB-OH (IC50 > 100 nmol/L) and the potent TUB-OMOM (IC50, ~1 nmol/L), and their direct covalent conjugation to (89)Zr-trastuzumab were established. Radioiodination of tubulysins was 92% to 98% efficient and conversion to N-hydroxysuccinimide (NHS) esters more than 99%; esters were isolated in an overall yield of 68% ± 5% with radiochemical purity of more than 99.5%. Conjugation of (131)I-tubulysin-NHS esters to (89)Zr-trastuzumab was 45% to 55% efficient, resulting in ADCs with 96% to 98% radiochemical purity after size-exclusion chromatography. ADCs were evaluated for their tumor-targeting potential and antitumor effects in nude mice with tumors that were sensitive or resistant to trastuzumab, using ado-trastuzumab emtansine as a reference. ADCs appeared stable in vivo. An average DAR of 2 and 4 conferred pharmacokinetics and tumor-targeting behavior similar to parental trastuzumab. Efficacy studies using single-dose TUB-OMOM-trastuzumab (DAR 4) showed dose-dependent antitumor effects, including complete tumor eradications in trastuzumab-sensitive tumors in vivo. TUB-OMOM-trastuzumab (60 mg/kg) displayed efficacy similar to ado-trastuzumab emtansine (15 mg/kg) yet more effective than trastuzumab. Our findings illustrate the potential of synthetic tubulysins in ADCs for cancer treatment.

Perspectives of combined radioimmunotherapy and anti‐EGFR antibody therapy for the treatment of residual head and neck cancer

Rhenium‐186 based radioimmunotherapy (RIT) may have potential for the treatment of minimal residual disease in patients with squamous cell carcinoma of head and neck (HNSCC). In an effort to enhance the efficacy of RIT, we evaluated the combination of RIT and anti‐epidermal growth factor receptor (EGFR) therapy in nude mice bearing established HNSCC s.c. xenografts. For this purpose we used the EGFR‐blocking monoclonal antibody (MAb) 425. Treatment of HNSCC‐bearing mice with the combination of a single administration of 200 μCi 186Re‐labeled MAb U36 as well as 1.1 mg unlabeled MAb 425 showed an enhanced efficacy in comparison to the single treatments. When 500 μCi 186Re‐labeled MAb U36 were administered, all tumors eventually regressed completely. The combination of this RIT treatment with multiple injections of MAb 425 significantly increased the rate of tumor regression. Although RIT with 186Re‐labeled MAbs appears to be very efficient on HNSCC xenografts, the combination with anti‐EGFR MAb 425 may enhance the efficacy. Int. J. Cancer 77:13–18, 1998.© 1998 Wiley‐Liss, Inc.

Functional Genetic Screens Identify Genes Essential for Tumor Cell Survival in Head and Neck and Lung Cancer

Purpose: Despite continuous improvement of treatment regimes, the mortality rates for non–small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC) remain disappointingly high and novel anticancer agents are urgently awaited. Experimental Design: We combined the data from genome-wide siRNA screens on tumor cell lethality in a lung and a head and neck cancer cell line. Results: We identified 71 target genes that seem essential for the survival of both cancer types. We identified a cluster of 20 genes that play an important role during G2–M phase transition, underlining the importance of this cell-cycle checkpoint for tumor cell survival. Five genes from this cluster (CKAP5, KPNB1, RAN, TPX2, and KIF11) were evaluated in more detail and have been shown to be essential for tumor cell survival in both tumor types, but most particularly in HNSCC. Phenotypes that were observed following siRNA-mediated knockdown of KIF11 (kinesin family member 11) were reproduced by inhibition of KIF11 using the small-molecule inhibitor ispinesib (SB-715992). We showed that ispinesib induces a G2 arrest, causes aberrant chromosome segregation, and induces cell death in HNSCC in vitro, whereas primary keratinocytes are less sensitive. Furthermore, growth of HNSCC cells engrafted in immunodeficient mice was significantly inhibited after ispinesib treatment. Conclusion: This study identified a wide array of druggable genes for both lung and head and neck cancer. In particular, multiple genes involved in the G2–M checkpoint were shown to be essential for tumor cell survival, indicating their potential as anticancer targets. Clin Cancer Res; 19(8); 1994–2003. ©2013 AACR.

Inert coupling of IRDye800CW and zirconium-89 to monoclonal antibodies for single- or dual-mode fluorescence and PET imaging

IRDye800CW and zirconium-89 (89Zr) have very attractive properties for optical imaging and positron emission tomography (PET) imaging, respectively. Here we describe a procedure for dual labeling of mAbs with IRDye800CW and 89Zr in a current good manufacturing practice (cGMP)-compliant way. IRDye800CW and 89Zr are coupled inertly, without impairment of immunoreactivity and pharmacokinetics of the mAb. Organ and whole-body distribution of the final product can be assessed by optical and PET imaging, respectively. For this purpose, a minimal amount of the chelate N-succinyldesferrioxamine (N-sucDf) is first conjugated to the mAb. Next, N-sucDf-mAb is conjugated with IRDye800CW, after which the N-sucDf-mAb-IRDye800CW is labeled with 89Zr. After each of these three steps, the product is purified by gel filtration. The sequence of this process avoids unnecessary radiation exposure to personnel and takes about 5 h. The process can be scaled up by the production of large batches of premodified mAbs that can be dispensed and stored until they are labeled with 89Zr.

A novel method to quantify IRDye800CW fluorescent antibody probes ex vivo in tissue distribution studies

BackgroundWe describe a new method for biodistribution studies with IRDye800CW fluorescent antibody probes. This method allows the quantification of the IRDye800CW fluorescent tracer in percentage of injected dose per gram of tissue (% ID/g), and it is herein compared to the generally used reference method that makes use of radioactivity.MethodsCetuximab was conjugated to both the near-infrared fluorophore IRDye800CW and/or the positron emitter 89-zirconium, which was injected in nude mice bearing A431 human tumor xenografts. Positron emission tomography (PET) and optical imaging were performed 24 h post-injection (p.i.). For the biodistribution study, organs and tumors were collected 24 h p.i., and each of these was halved. One half was used for the determination of probe uptake by radioactivity measurement. The other half was homogenized, and the content of the fluorescent probe was determined by extrapolation from a calibration curve made with the injected probe.ResultsTumors were clearly visualized with both modalities, and the calculated tumor-to-normal tissue ratios were very similar for optical and PET imaging: 3.31 ± 1.09 and 3.15 ± 0.99, respectively. Although some variations were observed in ex vivo analyses, tumor uptake was within the same range for IRDye800CW and gamma ray quantification: 15.07 ± 3.66% ID/g and 13.92 ± 2.59% ID/g, respectively.ConclusionsThe novel method for quantification of the optical tracer IRDye800CW gives similar results as the reference method of gamma ray quantification. This new method is considered very useful in the context of the preclinical development of IRDye800CW fluorescent probes for optical molecular imaging, likely contributing to the selection of lead compounds that are the most promising for clinical translation.